Co-existence of extended-spectrum β-lactamases blaCTX-M-9 and blaCTX-M-15 genes in Salmonella species isolated from febrile and diarrhoeagenic patients in Lagos, Nigeria: a cross-sectional study

Background Resistance to different antimicrobial classes by Salmonella species has generated a global public health concern. The spread of extended-spectrum β-lactamases (ESBLs) blaCTX gene variants is also increasing. This study aimed to investigate the antibiotic resistance and the carriage of blaCTX-M-9 and blaCTX-M-15 as well as the quinolone resistance gene (qnrB19) among Salmonella species from hospitalised patients in Lagos, Nigeria. Methods In this cross-sectional study from April 2021 to August 2021, a total of 508 samples were collected from hospitalised patients. The samples were subjected to standard microbiological investigation. All the isolates were identified using API 20E kits and real-time polymerase chain reaction (RT-PCR). The in vitro antibiotic susceptibility testing (AST) was investigated using the disk diffusion method. Detection of antibiotic resistance and virulence gene makers was conducted using RT-PCR. Results In total, 24 Salmonella species were identified. All the isolates were non-typhoidal Salmonella isolates. None of the isolates screened was S. Typhi and S. Paratyphi. Most of the isolates were susceptible to imipenem, ciprofloxacin, ofloxacin and gentamycin, while a high level of resistance to all cephalosporins, penicillin, and some carbapenems was observed. In total, 79.2% (19/24) of the Salmonella isolates harboured the blaCTX-M variant including 54.2% (13/24) blaCTX-M-9 and 12.5% (3/24) blaCTX-M-15, while co-habitation of blaCTX-M-9 and blaCTX-M-15 was observed in 12.5% (3/24) of the isolates, respectively. None of the isolates harboured quinolone-resistant qnrB19 gene and virulence gene stn. However, invA gene was present in 66.7% (16/24) of all isolates. Conclusions This study is considered the first report of blaCTX-M-9 and blaCTX-M-15 variants in Salmonella species in Nigeria. The continued existence of cefotaximase (CTX-M)-producing Salmonella within our environment calls for the prudent use of cephalosporins.


Background
Salmonella infections are recognised as a global public health challenge and are one of the most common causes of human gastroenteritis worldwide, sometimes with negative clinical outcomes especially among vulnerable individuals [1][2][3]. Invasive salmonellosis is presented in approximately 6% of patients with a clinical prognosis of diarrhoeal enterocolitis. Invasive non-typhoidal Salmonella (iNTS) is endemic especially in sub-Saharan Africa occurring in 79% of the estimated 535, 000 global cases of iNTS infection and 85% of the estimated 77, 500 death globally giving a 15.8% case fatality ratio [4,5].
Salmonella-associated infections are treated with ampicillin, chloramphenicol, and trimethoprim/sulfamethoxazole in the 1980s. However, the emergence of resistance clones to these antimicrobials resulted in the use of other antibiotic classes. Currently, several classes of antibiotics are available such as 3rd and 4th generation cephalosporin (cefpodoxime, ceftriaxone, ceftazidime, cefotaxime), fluoroquinolones (ciprofloxacin, moxifloxacin, levofloxacin) and carbapenems (ertapenem, doripenem, imipenem) which are essential in the treatment of salmonellosis with good therapeutic potentials. However, the misuse of these antibiotics classes in agricultural, animal and human medicine has been attributed to the emergence, persistence and eventual spread of resistant strains [6]. The continuous spread of multidrug-resistant bacterial pathogens has been attributed to prolonged hospitalisation causing increased morbidity and mortality [7].
Salmonella is increasingly becoming resistant from an estimated rate of 20-30% in the 1990s and increasing to over 30% a decade later [8]. There has been a global increase in the number of bacteria exhibiting resistance to third and fourth-generation cephalosporin, most times due to the production of extended-spectrum β-lactamases (ESBLs) and AmpC-type β-lactamases. Cefotaximase (CTX-M) type of ESBLs remains the widely distributed ESBL enzymes globally [9]. The prevalence rate of CTX-M-type β-lactamases differs among members of the family Enterobacteriaceae [10]. The earliest CTX-M-1 clone was detected in Germany in the late 1980s [11], consequently, an explosion of CTX-M-type β-lactamases was witnessed in the twenty-first century, with a report of CTX-M-2, 3,4,5,6,7,9,15 in South America, the Mediterranean and within the European countries [12].
The CTX-M-type β-lactamases belong to a novel Ambler molecular class A ESBL and are mostly plasmid-mediated with a preference for hydrolysing cefotaxime [13] with bla CTX-M - 15 being most prevalent [14]. In Nigeria, the first CTX-M-1 clone was reported in 2015 [15]. Incidentally, only bla CTX-M -1 , and bla CTX-M -3 clones have been documented by separate studies in Salmonella enterica [15,16]. So far, the report on CTX-M-9 and 15 have only been documented in Escherichia coli isolated from clinical and animal sources in Nigeria [17,18]. Eguale et al. [19] reported bla CTX-M - 15 in Salmonella from Ethiopia . Many other studies in Egypt have recorded both bla CTX-M-9 and bla CTX-M- 15 in E. coli [20,21]. The distribution of CTX-M type of ESBLs among Salmonella species is becoming a serious challenge to global health. The continued dissemination of this resistant clone among members of the family Enterobacteriales in Nigeria is also becoming worrisome. There is a paucity of reports on the CTX-M type of ESBLs among Salmonella serovars in Nigeria, especially from clinical sources. This study aimed to investigate the prevalence of Salmonella in febrile and diarrhoeal patients, their antibiotic resistance, and the carriage of bla CTX-M-9 and bla CTX-M- 15 ESBLs genes using real-time polymerase chain reaction (RT-PCR) in Lagos, Nigeria.

Ethics clearance
Ethical approvals from the Human Research and Ethics Committee of Lagos State University Teaching Hospital with reference number LREC/06/10/1012 and the Lagos State Health Service Commission with reference number LSHSC/2222/VOL.VC/352 were obtained prior to patients' enrolment in accordance with 1964 Helsinki declaration and its later amendments or comparable ethical standards. The written informed consent was obtained from all subjects and/or their legal guardians.

Study population, case definition and sample size
In this cross-sectional study, a total of 508 in-patients and out-patients who sought treatment at some public health care facilities in Lagos State, Nigeria, were recruited from April 2021 to August 2021. The patient's history and demography were obtained. Two categories of patients were recruited for this study: category A includes patients with febrile illness. Patients with febrile illness were defined as a patient whose body temperature is greater than (≥ 37.5 °C) in the last 3 days with one or more of the following symptoms: abdominal pain, loose stool, headache, vomiting, and/ or nausea, loss of appetite for up to 5 days. Category B includes patients with the diarrhoeal disease and was defined as a patient who had been diagnosed by a clinician with persistent loose stool for up to 72 h or more. The sample size was estimated using the formula for estimating the minimum sample for descriptive studies: N = Z 2 p (1-p)/d 2 , where, N = sample size, Z = 1.96 (standard error) at 95% confidence interval, p = prevalence of the disease as reported by Smith et al. [22] = 74%, and d = level of precision at 5% (0.05). Therefore, N = 295649536. Approximately 296 samples per 10,000 population. However, five hundred and eight (508) samples were collected to make the inference valid.

Collection and processing of samples
Blood samples (10 ml from adults and 5 ml from children 1-15 yrs) were aseptically obtained and inoculated into blood culture bottles containing brain heart infusion (BHI) broth (LAB M, Lancashire, UK) and were gently mixed. The bottles were incubated at 37 °C aerobically for 7 days. Turbid bottles were sub-cultured on xylose lysine agar (XLD) (HIMEDIA, Mumbai, India), MacConkey agar (HIMEDIA, Mumbai, India), and Salmonella-Shigella Agar (SSA) (HIMEDIA, Mumbai, India) and were further incubated for 18-24 h at 37 °C. Non-turbid blood culture bottles were also subcultured and repeated daily from the BHI broth for up to 7 days, after which the blood-broth suspension was finally discarded. A loop full of stool samples was inoculated into 10 ml of alkaline peptone water (HIMEDIA, Mumbai, India) for pre-enrichment. The suspension was incubated for 18 h at 37 °C. One ml of the bacterial suspension was dispensed into 9 ml of Selenite F broth (HIMEDIA, Mumbai, India), and the suspension was incubated for at 37 °C for 18 to 24 h. After incubation, a loop full of the bacteria suspension was inoculated into MacConkey agar (HIMEDIA, Mumbai, India), blood agar (LAB M, Lancashire, UK), bismuth sulphite agar (BSA) (LAB M, Lancashire, UK), Salmonella-Shigella agar (SSA) (HIMEDIA, Mumbai, India), and xylose lysine agar (XLD) (HIMEDIA, Mumbai, India). The media were incubated at 37 °C for 18 to 24 h.

Bacterial identification
Bacteria identification was carried out using the analytical profile index (API) 20E identification system (Institut Mérieux, Marcy l'Etoile, France) according to the manufacturer's instructions. The results of several biochemical parameters were used to generate a profile index code. The profile index code generated was used for the identification of all the isolates in the API-WEB database (https:// apiweb. biome rieux. com).

Extended-spectrum β-lactamase (ESBL) assay
All Salmonella isolates with reduced susceptibility and/or resistance to 3rd generation cephalosporin (3GCs) were screened for ESBL production phenotypically using the double disk synergy test (DDST) method as previously described [16]. Escherichia coli ATCC ® 25922 ™ was used as negative control while Klebsiella pneumoniae ATCC ® 700603 ™ was used as a positive control.

DNA extraction from bacterial isolate
The DNA extraction was carried out using a QIAamp DNA mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions as described by Akinyemi et al. [16].

RT-PCR
The RT-PCR Rotor gene Q 2plex (Qiagen, Hilden, Germany) was used for the amplification and detection of STY0313/SPA2475/t2576 and STY0316/t2574 for Salmonella Typhi and Salmonella Paratyphi A. Salmonella virulence genes invA, stn, and antibiotics resistance genes bla CTX-M-9 , bla CTX-M-15 , and qnrB19 were screened using the TaqMan technology. The following previously published oligonucleotides sequences specific for detection of invA, stn, bla CTX-M-9 , bla CTX-M-15 , and qnrB19 were used [16,[24][25][26][27] (Table 1). The qPCR assays were performed in a Qiagen rotor gene Q 2plex thermocycler (Qiagen, Hilden, Germany) with 72 well reaction tubes, closed with 4-Cap Strips. The reaction was performed by dispensing 12.5 μl of 2 × SYBR ® Green Taq PCR master  [16]. The threshold limit setting was performed in automatic mode. A "no template" control (NTC) was included in each reaction. Only signals generated in SYBR ® Green qPCR analysis that displayed amplification above the threshold level were considered positive. While those signals with no C t value obtained were considered negative.

Statistical analysis
These data generated were entered into Microsoft Excel spreadsheet 2010 and were analysed using Statistical Package for Social Sciences (SPSS) Windows (Version 15.2; Chicago, IL) and Epi Info (Version 6.0, USD, Stone Mountain, GA). Prevalence of Salmonella and its association with demographic determinants among febrile and diarrhoeagenic patients in selected health care facilities were determined. The prevalence of salmonellosis was calculated as the number of positive cultures for Salmonella divided by the total number of samples collected. The data were interpreted using the Chi-square test of independence, while the strength of association was determined by estimating the odds ratio (OR). The statistical test was considered significant if the P-value was ≤ 0.05. A correlation analysis was also performed to determine the association of virulence genes and antimicrobial resistance genes belonging to the same antimicrobial class or different classes among the investigated Salmonella isolates. Binary data (0) indicating absent and (1) indicating presence of virulence and resistance genes were imported into an R software (version 4.1.3 and RStudio IDE. https:// www.r-proje ct. org; https:// www. rstud io. com. Accessed, 3 September 2022). The correlations were then calculated with package "ggcorrplot" using the function cor.mtest" at a significance of P < 0.05.

Demographic distribution of Salmonella isolates
The proportion of Salmonella species was 6.3% (15/240) in males and 3.4% (9/268) in females. Out of the 24 Salmonella species detected, 15/24 representing 62.5%, were isolated from children between the ages of 1-10 years, with a mean age of 4.7 years. Also, 25.0% (6/24) of the total Salmonella isolates were detected from patients between the ages of 11-20 years, with a mean age of 14.3 years. Meanwhile, the remaining 12.5% (3/24) was isolated from patients within the age bracket 21-30 years with a mean age of 24.7 years (Tables 2 and 3). The prevalence of Salmonella-associated bacteraemia stood at 3.9% Table 1 Primers with nucleotide sequence use in the study

Antibiotic susceptibility and pattern of resistance
The result of the in vitro antimicrobial susceptibility testing showed some level of susceptibility to imipenem (75.0%), ciprofloxacin (62.5%), gentamycin (62.5%) and ofloxacin (50.0%), while all the isolates developed resistance (100.0%) to ceftazidime, cefotaxime, cefoxitin, augmentin, ampicillin, ertapenem, and doripenem. However, 75.0% of the isolates showed resistance to cefpodoxime. Resistance to ofloxacin and amikacin was 50.0% each. All Salmonella isolates were multiple drug resistant (MDR). MDR was defined as resistance to at least one agent in three or more antimicrobial categories (Table 4). All the isolates developed resistance to between 7 and 13 antibi-  (Table 5). High multiple antibiotic resistance (MAR) index (ranging from 0.5 to 1.0) was observed for all the Salmonella isolates. All the Salmonella isolates from this study were negative when screened for Salmonella Typhi and Salmonella Paratyphi A gene using STY0313/ SPA2475/t2576-F and STY0316/t2574-R primer pairs (Table 6).

Virulence and CTX-M genes
Sixteen of the isolates (66.7%) harboured the invasive (invA) gene, while none of the isolates harboured the enterotoxin (stn) gene. In total, 79.2% (19/24) of the Salmonella isolates harboured the bla CTX-M variant including 52.0% (13/24) bla CTX-M-9 and 12.5% (3/24) bla CTX-M-15 , respectively. Six Salmonella isolates from diarrhoeal patients and seven Salmonella isolates from patients with febrile illness harboured bla CTX-M-9 only . Two Salmonella isolates from febrile patients and one from diarrhoeal patients harboured bla CTX-M-15 only, respectively. Of note was the co-existence of bla CTX-M-9 and bla CTX-M-15 in 3 (12.5%) Salmonella isolates from febrile patients with similar resistance pattern CAZ-CPD-CTX-FOX-AUG-AMP-ETR-DOR. All the isolates were negative for the plasmid-mediated quinolone resistance gene qnrB19 (Table 6).

Correlation analysis
The result of the correlation analysis revealed a significant association between the invA gene and bla CTX-M-9 and a strong association between bla CTM-9 and bla CTX-M-15. Significance was calculated at P < 0.05, and boxes with non-significant correlations were left blank as such genes were not detected (Fig. 1).

Discussion
Salmonella-associated infection caused by the genus Salmonella remains a public health challenge in Africa. Salmonella has been associated with an increasing number of reported cases of community-acquired bloodstream infections, self-limiting gastroenteritis to more severe infections resulting in a high rate of morbidity and mortality [16]. Fluoroquinolone and extendedspectrum cephalosporin are used as first-line antibiotics for the treatment of salmonellosis. However, treatment failure due to resistance to these classes of antibiotics has resulted in prolonged hospitalisation with negative clinical outcomes [7]. In this study, 24 Salmonella species were detected from 508 samples with an overall prevalence of 4.7%. The prevalence of Salmonella-associated bacteraemia was 3.9%, while Salmonella-associated gastroenteritis was 6.9%. This result was similar to 3.9% prevalence of Salmonella-associated bacteraemia reported in Nigeria by Akinyemi et al. [16], but at variance with the 1.9% recorded by the same author in a separate study [28]. All the Salmonella species detected in this study were non-typhoidal Salmonella because none of the primer pairs used for the detection of Salmonella Typhi and Salmonella Paratyphi A yielded Ct value. The Prevalence of Salmonella-associated gastroenteritis (SAG) in this study was 6.9%. The result was lower when compared to the results of similar studies; 9.6%, 16.3% and 16.6% that were conducted in different periods in 2021, 2018 and 2007 in Lagos, Nigeria [16,29,30], respectively. However, this study was consistent with the 6.2% (59/957) reported in Ethiopia [31]. Conversely, lower prevalence of 1.3% and 2.7% were reported in separate studies in Ethiopia [32,33], 3.0% in Kenya [34] and 3.5% in Egypt [35] compared to 6.9% SAG reported in this study. Also, the 6.9% SAG recorded in this study was lower when compared to the 13.80% in Tanzania [36] 17.86% in Thailand [37] and 20.39% in China [38]. The observed variations in the prevalence of Salmonella infection in this study when compared to other studies, could be attributed to various factors such as sample size, gender, and age distribution, location of sampling and seasonal variation [36]. Fifteen out of the 20 (15/24) Salmonella species detected representing 62.5% were isolated from children between the ages of 1-10 years, with a mean age of 4.7 years, standard deviation (SD) ± 2.859, margin of error (ME) ± 0.2517 (4.7364 ± 0.494 (± 10.42%) at 95% confidence level. The prevalence of Salmonella infection in this study was higher among children < 10 years when compared to other age groups. Children have been identified as a vulnerable population group at risk of Salmonella infection due to underdeveloped immune system and exhibition of poor hygienic habits [38]. Report on high prevalence of Salmonella infection in children is not uncommon. Several studies have documented a high prevalence of Salmonella in children in different countries such as Nigeria [16,39], China [38,40], and Italy [41]. There were variations in the observed clinical presentations of the patients in this study and intravenous saline (0.8%) was administered in those patients with severe dehydration. The result of the in vitro antimicrobial susceptibility testing showed that all the 24 Salmonella enterica detected were MDR as they were resistant to three or more classes of antibiotics. Notably, 100% resistance to all the following β-lactam antibiotics (ceftazidime, cefotaxime, cefoxitin, augmentin and ampicillin) and carbapenems (ertapenem and doripenem) were observed in all the Salmonella species, while 75% (18/24) Salmonella isolates showed resistance to cefpodoxime. Regrettably, in Nigeria, similar level of resistance was reported by other studies [16,42,43]. Studies conducted elsewhere recorded similar resistance in Salmonella species. In Ethiopia, 88.9% of all the isolates were resistant to ampicillin [28], 88% in Bangladesh [40] and 74% resistance to cefotaxime was reported by Harakeh et al. [44] in Lebanon. High-level resistance to β-lactam antibiotics was also reported in Burkina Faso [45], Canada [46], and the USA [47]. The high level of resistance among Salmonella to β-lactam antibiotics and carbapenems as revealed in this study is worrisome and calls for urgent attention. The differences in the percentage of resistance from different countries may be due to different risk factors such as Table 4 Antibiogram of non-typhoidal Salmonella isolates from clinical samples by disc diffusion method sickle cell disease, HIV, malnutrition, tuberculosis and other respiratory infections, all of which are conditions known to be associated with frequent antibiotic use [39]. Reduced susceptibility to imipenem (25.0%), ciprofloxacin (37.5%), ofloxacin (50.0%), gentamycin (37.5%), and amikacin (50.0%) was recorded in this study. Reduced susceptibility to fluoroquinolones is a serious public health concern, as this class of antibiotics remains among the antimicrobials of choice for the treatment of invasive and systemic salmonellosis in human medicine and animals' husbandry [48,49]. This study revealed high multiple antibiotic resistance (MAR) index (ranging from 0  from the clinical samples have been reported in Iran [52]. Ethiopia [19], India [9] Zambia [53], Kuwait [54], Japan [53], France and Senegal [25], and Germany [55]. Therefore, this study has demonstrated the potential dissemination of bla CTX-M variants in Salmonellae from our environment with a prolonged hospital stay of patients and consequently resulting in treatment failure. Resistance to this class of antibiotic by Salmonella has generated global public health concern [56], and this is now a major concern in Nigeria as 50% of the patients harbouring Salmonella with CTX-M genes in this study are children < 10 years of age. There have been successful global dissemination of CTX-M genes, two of these most common variant bla CTX-M-9 and bla CTX-M-15 were detected in this study. Although, in Nigeria, bla CTX-M-9 and bla CTX-M-15 have been reported in E. coli isolated from stool samples of pregnant women [17] and from animal sources [18]. To the best of our knowledge, this is the first report of bla CTX-M-9 and bla CTX-M-15 in Salmonella isolated from clinical sources in Nigeria. In this study, out of the bla CTX-M-9 and bla CTX-M-15 -producing Salmonella strains detected, 50% (12/24) were simultaneously resistant to ciprofloxacin and ofloxacin in vitro. However, none of these isolates harboured plasmid-mediated quinoloneresistant gene qnrB19 genes. This gene if present would have enhance the development of quinolone resistance strains [57]. None of the Salmonella isolates from this study harboured the enterotoxin (stn) gene. The expression of this gene is responsible for Salmonella-associated gastroenteritis by inducingpreviously been reported in Nigeria [59] and Egypt [60]. However, the detection of invasive (invA) gene which is a common molecular target for Salmonella-specific detection was recorded in 66.7% (16/24) of Salmonella species investigated, an indication that qPCR investigation of invA gene is a suitable target for the confirmation of putative Salmonella isolates. The invA gene of Salmonella species is associated with invasion of host epithelial cell and it is located on the pathogenicity island 1 (SPI-1). The detection of this virulence gene in Salmonella as a diagnostic application had been reported by several authors with varying degree of sensitivity and specificity ranging from 55 to 100% [16,61,62].

Conclusion
This study revealed prevalence of 4.7% Salmonellaassociated infections, with 62.5% of the Salmonella isolates detected found in children ≤ 10 years. All Salmonella isolated exhibited high level of resistance to β-lactams antibiotics and carbapenems with reduced susceptibility to fluoroquinolone. The study revealed for the first time the carriage of bla CTX-M-9 and bla CTX-M-15 variants among the Salmonella species from Nigeria. Complete therapeutic failure of first-line antibiotics is eminent, if the policy-makers do not only ensure a complete barn on the over-the-counter-sale of antibiotics without doctor's prescription, but also a routine follow-up programme for enforcement, to curb the spread of antibiotics resistance organisms.